Project Summary A fundamental gap exists in understanding the mechanisms that protect oocytes during aging and during stress. The maintenance of oocyte quality during stress and aging is positively correlated with the assembly of large granules composed of mRNA and RNA-binding proteins, termed RNP granules. However, neither the regulation of RNP granule assembly, nor the function of RNP granules in vivo are well understood. The long-term goal is to identify the mechanisms that allow the germ line to adapt and maintain its integrity during the prolonged meiotic arrest that occurs during aging, and during stress. The primary objective of this application is to identify the molecular mechanisms that modulate RNP granule dynamics and function in stressed and meiotically-arrested germ lines. The two central hypotheses of this proposal are: 1) alterations in chaperone protein function and decreased MAP Kinase activity, trigger the formation of RNP granules in meiotically-arrested oocytes that function to maintain oocyte quality and repress maternal mRNAs, and 2) these triggers are distinct from those promoting the formation of RNP granules in stressed oocytes. These hypotheses have been formulated based on the results from a genetic screen and the characterization of RNP granule formation as an adaptation to osmotic stress completed in the PI?s lab. The overall approach is to investigate germ line RNP granule dynamics and function using the nematode C. elegans. The approach is innovative because it takes advantage of a system with naturally- occurring RNP granule assembly and dissociation in oocytes of a multicellular animal. It is also conceptually innovative in regards to involving undergraduate and master?s students in meritorious research aimed at understanding the role of chaperone proteins in the function and regulation of RNP granules during C. elegans oogenesis. The first aim is to characterize the specificity of chaperone proteins in regulating diverse RNP granules, and their function in oocytes. The roles of TriC, Hsp40, and Hsp70 chaperone proteins in RNP granule assembly will be characterized, as well as their roles in maintaining oocyte quality and regulating translation of maternal mRNAs. The second aim is to investigate the role of MAP Kinase phosphorylation in regulating chaperones and the assembly of RNP granules. The effects of altering levels of MAP Kinase activity on the assembly of RNP granule proteins, that are known MAPK substrates, will be determined. In vitro kinase assays will be performed to determine if the TriC candidate substrates are phosphorylated by MAP Kinase, and phosphorylation sites will be mapped in order to test their role in modulating RNP granule dynamics. This project is significant as it will uniquely contribute to the field by elucidating the roles of chaperone proteins and MAP kinase phosphorylation in modulating RNP granule assembly, and the oogenic roles of chaperone proteins.